Identification of Mesenchymal Stem Cell Marker STRO-1 in Oral Reactive Lesions by Immunofluorescence Method.

Statement of the Problem Stem cells are considered as new implement for tissue regeneration. Several niches in adult human body are colonized by multipotent stem cells but access to these potential reservoirs is often limited. Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is still unknown whether stem cells also exist in reactive lesions of oral cavity such as pyogenic granuloma and peripheral ossifying fibroma which are deliberated as inflammatory proliferation of different cell families. Purpose The aim of this study was to explore for clues to see whether pyogenic granuloma or peripheral ossifying fibroma contain dental mesenchymal stem cell (DMSC). Materials and Method Four pyogenic granuloma and four peripheral ossifying fibroma specimens were collected by excisional biopsy and preserved in PBS-EDTA at -86 °C. Then we cut them in 5µm diameter using Cryostat. Having been rinsed with PBS, the samples were stained with a primary mouse anti-human STRO-1 monoclonal IgM antibody. Afterward, a secondary goat anti-mouse IgM-FITC antibody was applied to detect STRO-1+ cells as probable stem cells by immunofluorescence technique. Results Immunofluorescence microscopy revealed presence of STRO-1+ cells in these lesions, particularly localized on perivascular zone. The negative control group was not glowing. Conclusion Based on these results, it was found that reactive lesions of pyogenic granuloma and peripheral ossifying fibroma have STRO-1 positive cells, which raises the possibility that these cells may be DMSCs.


Introduction
Stem cells are promising tools for tissue regeneration.
Their remarkable ability in proliferation and differentiation enables them to restore the structures of impaired tissues. [1] The need to generate variety of tissues as well as the restriction of differentiation potential of stem cells after birth (compared with embryonic stem cells) have led to discovery of various resources of adult stem cells. [2] Various places in human body are colonized by considerable number of these cells, however, since access to these areas by surgery may cause damages to them, surgery would be considered as restricting factor in using these cells. For example, collecting these cells from the CNS causes deficiencies and problems that put the benefit of using these cells into question. On the other side, the selected locations should have a high percentage of stem cells compared with the amount of removed tissue.
The application of adult stem cells (ASCs) in tissue engineering is not followed by legal and ethical issues. Recently, most of ASCs are multipotent mesenchymal stromal cells (MSCs) [2] that have the capability of being transformed to different cells such as osteoblasts, [3] hepatocytes, [4] neurons, [5] adipocytes, cementoblasts, odontoblasts, [6] and cardiomyocytes. [7] These cells can make specific lesions heal through secretion of anti-inflammatory and nutrient materials.
The major source of ASCs is the bone marrow; however, they can be obtained from other reservoirs such as adipose tissue [1] and dental pulp. Dental pulp and its supportive tissues are derived from a sort of tissue called ectomesenchyme, which is produced by interaction of neural crest cells and mesenchyme in embryonic period. Hence, dental stem cells are likely to have properties identical to mesenchymal stem cells such as those of bone marrow. Despite the similarities, these two cell groups have some differences. For instance, stem cells of dental tissue are more differentiated, undergo fewer changes, and can help more in development and progression of odontogenic rather than osteogenic differentiation. [11] In recent years, mesenchymal stem cells with high proliferative ability have been isolated from dental ectomesenchymal tissues such as periodontal ligament stem cells (PLSCs) [12] and dental pulp stem cells (DPSCs). [8][9] Moreover, based on a study, the DPSCs have been detected in hyperplastic pulpitis (pulp polyp), [13] since pulp polyp is characterized as a reactive lesion of oral cavity; it is likely to happen to other reactive lesions of oral cavity. Pyogenic granuloma is one of these lesions that are histopathologically similar to pulp polyp. Peripheral ossifying fibroma is another reac-tive lesion of oral cavity with a similar pathogenesis. To remove these lesions completely, both lesions are treated by excisional surgery. STRO-1 is a cell marker present on all clonogenic stromal precursors [14] and is most commonly used in stem cell researches as a reliable marker for mesenchymal stem cells. [15][16] The aim of this study was to find clues on presence of dental mesenchymal stem cell (DMSCs) in these lesions, using an immunofluorescent technique to detect DMSC marker STRO-1.

Materials and Method
Eight patients with pyogenic granuloma and peripheral ossifying fibroma who had referred to Shiraz School of Dentistry were examined and their diagnosis was con-

Results
In this study, four pyogenic granulomas and four peripheral ossifying fibromas were studied. Table 1 represents the patients' demographic information. As-   DMSCs are capable of forming dentin and pulp-like complex, and it makes the multipotent cells to be used as a perfect source for regenerating pulp and dental tissues. [23] Since the first researches, all types of dental mesenchymal stem cells have represented the ability to generate mineralized nodules with high levels of calcium when being placed in osteogenic culture medium, [15,17,[24][25]] and the researchers have tested and confirmed their differentiation ability.
Cryoprotection of cells and tissues, especially in repair processes, has significantly improved recently. [26] Up to now, only hematopoietic stem cells have been protected by cryoprotection [27][28] and since then,